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NEWS AND VIEWS
RIGing a virus trap
Chris A Benedict & Carl F Ware
A new player in the innate defense system
has recently emerged, RIG-I (retinoic acidinducible gene I). RIG-I recognizes the RNA
of RNA viruses and has a more famous counterpart in innate immune defense, the Toll-like
receptors (TLRs), which also recognize conserved molecular components of pathogens.
RIG-I operates differently than the TLRs—for
instance, RIG-I functions in the cytoplasm,
whereas TLRs function at the cell surface or in
endosomal compartments. But both TLR and
RIG-I signaling culminate in the induction of
the type I interferon (IFN-α/β) response—one
of the earliest alarm bells of the immune system, which counteracts viral replication without killing the infected cell.
In a recent issue of Immunity, Kato et al.
report that RIG-I induces IFN-α/β in several
cell types infected with RNA viruses, but TLR
signaling is dominant in plasmacytoid dendritic cells, highly specialized cells that make
extraordinarily high levels of IFN-α/β1. Earlier
this year, two reports2,3 showed that hepatitis C virus (HCV) targets the RIG-I pathway,
thereby promoting virus replication. Together,
these results place RIG-I at a crucial position
in the innate response to viral infection, and
highlight the diverse pathways used to recognize pathogen-specific molecules.
RIG-I consists of two caspase recruitment
(CARD) domains and an RNA-binding helicase
domain, and is one several proteins consisting of
these fused domains. CARD domains are present in various proteins involved in proinflammatory or cell-death pathways. Previous work
has shown that RIG-I recognizes viral doublestranded RNA through its helicase domain, and
induces downstream signals leading to IFN-α/β
production through its CARD domains4.
To assess the importance of RIG-I in
regulating the IFN-α/β response, Kato et al.
generated RIG-I–deficient mice1. Most of
these mice died a mysterious death during
Chris A. Benedict and Carl F. Ware are in the
Division of Molecular Immunology, La Jolla Institute
for Allergy and Immunology, San Diego, California
92121, USA.
e-mail: [email protected] or [email protected]
RNA virus
Helicase
CARD CARD
Viral
dsRNA
TLR7/8
IRF7
88
MyD
IRAK4
RIG-I
Viral
ssRNA
HCV
NS3/4a
?
Endosome
TRAF6
IRF5
TBK1, IKKε
NF-κB
JNK
p38
IRF7
IRF3
IFN-β
IFN-β
IFN-α
Katie Ris
© 2005 Nature Publishing Group http://www.nature.com/naturemedicine
Molecules that recognize pathogens and activate the immune response are being discovered at a rapid rate. RIG-I,
a new protein in this category, recognizes viral RNA. Recent studies show that RIG-I operates independently of
Toll-like receptors and that it is targeted for inactivation by the hepatitis C virus.
IL-6
IL-12
Hepatocyte
(fibroblast, conventional dendritic cell)
Plasmacytoid dendritic cell
Figure 1 TLR- and RIG-I–dependent induction of type I interferon during RNA virus infection. In
plasmacytoid dendritic cells, the interaction of viral single-stranded RNA (ssRNA) with TLR7 (TLR7
or TLR8 in humans) probably occurs in an early endosomal compartment after virus uptake into the
cell. The cytoplasmic adaptor protein MyD88 and interferon response factor-7 (IRF7) are required for
downstream induction of IFN-α/β gene expression. Expression of additional inflammatory cytokines,
such as interleukin (IL)-6 and IL-12, is also dependent upon MyD88, but this pathway uses the adaptor
proteins TNF receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinase 4 (IRAK4) and
IRF5. In other cell types (right panel) cytosol-localized RIG-I recognizes viral RNA, such as found in
hepatitis C virus. RIG-I uses a C-terminal RNA-helicase domain to bind RNA with double-stranded
structure, and transduces downstream signals leading to IFN-β transcription through two N-terminal
caspase recruitment domains (CARD). The HCV nonstructural 3/4a (NS3/4a) protease inhibits RIG-I
induction of IFN-β, but does not cleave RIG-I or the downstream IKKε and TBK1 kinases directly,
indicating the existence of an as yet unidentified protein targeted by NS3/4a.
embryogenesis due to massive liver apoptosis,
but a few offspring survived to several weeks
of age, allowing for analysis of their cells in
culture. Fibroblasts derived from embryos of
RIG-I–deficient mice mounted a poor IFN-β
response when infected with different RNA
viruses, and did not activate transcription
factors that mediate this response, such as
NF-κB and interferon response factor (IRF)-3.
Vesicular stomatitis virus, a negative-strand
RNA virus, replicated to higher levels in
RIG-I–deficient fibroblasts. Treatment of
RIG-I–deficient cells with IFN-β restored
resistance, confirming a role for RIG-I in the
NATURE MEDICINE VOLUME 11 | NUMBER 9 | SEPTEMBER 2005
induction of IFN-β not in its downstream
antiviral effects.
The authors then examined the induction
of IFN-α/β by a distinct RNA-based virus,
Newcastle disease virus (NDV), in dendritic
cells. There are several subsets of dendritic
cells: some capture and present antigen to
T lymphocytes (conventional dendritic cells)
and others are known for their robust production of IFN-α/β (plasmacytoid dendritic
cells). Conventional dendritic cells from
the spleen or derived from bone marrow of
RIG-I– deficient mice produced almost no
IFN-α/β when infected with NDV. In contrast,
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IFN-α/β production by RIG-I–deficient plasmacytoid dendritic cells was essentially normal
after NDV infection. In these cells, TLR instead
mediated the induction of IFN-α/β.
Thus, RIG-I seems to induce IFN-α/β production in several cell types, but TLRs remain the key
pathway for innate recognition of RNA viruses in
plasmacytoid dendritic cells. The use of these two
pathways in different cell types may reflect adaptation of host defense to different pathogens.
Earlier this year, Foy et al. and Breiman
et al. found that HCV has developed a mechanism to counteract RIG-I2,3. They traced
this mechanism to the HCV nonstructural
protease 3/4a (NS3/4a). When expressed in
cell cultures with RIG-I, NS3/4a could prevent RIG-I from inducing IFN-β.
Breiman et al. showed that overexpression of
the cytosolic kinases IKKε or TBK1 involved in
the activation of IFN-β could circumvent the
NS3/4a-mediated block in IFN-β induction.
Both groups concluded that RIG-I is not a
proteolytic substrate for NS3/4a, placing the
HCV block somewhere between RIG-I and
IKKε, perhaps acting upon an unidentified
adaptor protein (Fig. 1).
Foy et al. went on to harness this information to generate a small peptide that inhibits
the active site of the NS3/4a protease. The peptide inhibitor enhanced the IFN-β response
and decreased HCV replication in cell culture.
Similar inhibition of HCV replication was
observed in cells that overexpressed IKKε3.
These results suggest that antagonizing
NS3/4a proteolytic activity in HCV infected
individuals may increase the antiviral immune
response, hopefully limiting the ability of this
virus to establish a persistent infection.
Over the last few years, TLR-dependent signaling pathways have stood in the spotlight
of innate immunity, and deservedly so, given
the importance and diversity of this system in
pathogen recognition. The characterization of
multiple receptors and cytoplasmic adaptor
proteins required for TLR signaling has facilitated the discovery of additional pathways, such
as RIG-I, which function independently of TLR
but use overlapping cytoplasmic signaling cascades. RIG-I, for instance, operates in the cell
cytoplasm, whereas TLRs are localized at the
cell surface or in endosomal compartments.
The plasmacytoid dendritic cell, or natural
IFN-producing cell as it is known in humans,
appears to be the major IFN-α/β–producing
cell type. This may be because of the high level
of expression of TLR7 and TLR9 and constitutive expression of IRF7, a downstream effector necessary for the type I IFN response5.
Additionally, plasmacytoid dendritic cells
retain TLR ligands for extended periods of
time in early endosomal compartments, perhaps allowing for sustained signaling6.
So why don’t all cell types have the capability to respond with the vigor of a plasmacytoid dendritic cell when confronted with
an invading pathogen? Perhaps, more is not
always better. A growing body of evidence
indicates that dysregulated IFN-α/β production leads to autoimmune disorders, a price
paid for too vigorous of a response7.
This ‘new’ class of RNA helicase-CARD domain
proteins shows functional diversity, as indicated
by studies of a homolog of mouse RIG-I known
as Helicard. Helicard operates during apoptosis, is
a substrate for caspases, and increases DNA degradation through its helicase domain8. Another
link between cell-death pathways and the innate
response to virus is that the Fas-associated death
domain–containing protein is required in some
cells for the induction of IFN-α/β by doublestranded RNA9. It is unclear what regulates the
balance between cell survival and death pathways
by this family of helicase-CARD proteins.
The emergence of RIG-I as a conduit between
recognition of viral nucleic acid and the innate
immune response adds insight into how these
various signaling pathways interconnect.
More practically, these results provide a new
opportunity to modulate the interferon pathway
in individuals infected with HCV or other
pathogens10,11.
1. Kato, H. et al. Immunity 23, 19–28 (2005).
2. Foy, E. et al. Proc. Natl. Acad. Sci. USA 102, 2986–
29891 (2005).
3. Breiman, A. et al. J. Virol. 79, 3969–3978 (2005).
4. Yoneyama, M. et al. Nat. Immunol. 5, 730–737
(2004).
5. Liu, Y.J. Annu. Rev. Immunol. 23, 275–306 (2005).
6. Honda, K. et al. Nature 434, 1035–1040 (2005).
7. Theofilopoulos, A.N., Baccala, R., Beutler, B. & Kono,
D.H. Annu. Rev. Immunol. 23, 307–336 (2005).
8. Kovacsovics, M. et al. Curr. Biol. 12, 838–843
(2002).
9. Balachandran, S., Thomas, E. & Barber, G.N. Nature
432, 401–405 (2004).
10. Zhong, J. et al. Proc. Natl. Acad. Sci. USA 102, 9294–
9299 (2005).
11. Sumpter, R. Jr. et al. J. Virol. 79, 2689–2699
(2005).
Hatching a drug
Human monoclonal antibodies have hatched from chicken eggs
(Nat. Biotechnol. 23, 1159–1169). Generating antibodies in
eggs could prove more efficient than more traditional methods
of cell culture.
To generate antibody-producing eggs, Lei Zhu et al.
created an antibody transgene hooked up to regulatory elements
for albumin, which is expressed at high levels in eggs. The
investigators then transfected that gene into chicken embryonic
stem cells; these cells, in turn, were used to create chickens
that pumped out antibodies in the tubular gland cells of the
oviduct (cells derived from stem cells are labeled in green
(GFP), antibody in red and DNA in blue).
The egg-derived antibodies largely live up to the quality
standards of antibodies made in cultured cells—having, for
instance, similar affinity to antigen. Some 17 monoclonal
antibody–based drugs have been approved by the US Food
and Drug Administration in the last 20 years and many more
are on deck.
Charlotte Schubert
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Courtesy of Origen Therapeutics
© 2005 Nature Publishing Group http://www.nature.com/naturemedicine
NEWS AND VIEWS
VOLUME 11 | NUMBER 9 | SEPTEMBER 2005 NATURE MEDICINE